Homeodomain leucine zipper (HDZip) proteins constitute a family of transcription factors characterized by the presence of a DNA-binding domain (HD) and an adjacent leucine zipper (Zip) motif. The homeodomain usually consists of 60 conserved amino acid residues that form a helix1-loop-helix2-turn-helix3 that binds DNA. This DNA binding site is usually pseudopalindromic. The leucine zipper, adjacent to the C-terminal end of the homeodomain, consists of several heptad repeats (at least four) in which usually a leucine (occasionally a valine or an isoleucine) appears every seventh amino acid. The leucine zipper is important for protein dimerisation. This dimerisation is a prerequisite for DNA binding (Sessa et al. (1993) EMBO J 12(9): 3507-3517), and may proceed between two identical HDZip proteins (homodimer) or between two different HDZip proteins (heterodimer).
Homeodomain genes are present in all eucaryotes, and constitute a gene family of at least 89 members in Arabidopsis thaliana. The leucine zipper is also found by itself in eucaryotes other than plants. However, the presence of both a homeodomain and a leucine zipper is plant-specific (found in at least 47 out of the 89 proteins in Arabidopsis), and has been encountered in moss in addition to vascular plants (Sakakibara et al. (2001) Mol Biol Evol 18(4): 491-502). The leucine zipper is then located at the C-terminal end of the homeodomain, these two features overlapping by three amino acids.
The Arabidopsis HDZip genes have been classified into four different classes, HDZip I to IV, based on sequence similarity criteria (Sessa et al. (1994) In Plant Molec Biol, pp 412-426). Like the HD-Zip proteins from the three other classes, class I HDZip proteins are quite divergent in their primary amino structure outside of the homeodomain and the leucine zipper. Within both the homeodomain and the leucine zipper, class I HDZip proteins are further characterized by two specific features:                1) in the homeodomain, in addition to the invariant amino acids Leu16Trp48Phe49Asn51Arg53, position 46 is occupied by an Ala (A) and position 56 by a Try (W) (or occasionally by a Phe (F)) (Sessa et al. (1997) J Mol Biol 274(3):303-309; see FIG. 1), referred to as a class I homeodomain, and            2) the leucine zipper comprises six heptads, except for the fern Ceratopteris richardii which presents seven heptads (within each heptad, positions are named a, b, c, d, e, f and g, the conserved leucine being at position d; Sakakibara et al. (2001) Mol Biol Evol 18(4): 491-502; see FIG. 1). HDZip II, III and IV present a leucine zipper with five heptads only.
Concerning their DNA binding properties, class I HDZip proteins preferably bind to 5 bp half-sites that overlap at a central position, CAA(A/T)ATTG (Sessa et al. (1993) EMBO J 12(9): 3507-3517).
Different HDZip proteins have been shown to either activate or repress transcription. In Arabidopsis, the class I HDZip ATHB1, -5, -6, and -16 were shown to act as transcriptional activators in transient expression assays on Arabidopsis leaves using a reporter gene (luciferase; Henriksson et al. (2005) Plant Phys 139: 509-518). Two rice class I HDZip proteins, Oshox4 and Oshox5, acted as activators in transient expression assays on rice cell suspension cultures using another reporter gene (glucuronidase; Meijer et al. (2000) Mol Gen Genet 263:12-21). In contrast, two rice class II HDZip proteins, Oshox1 and Oshox3, acted as transcriptional repressors in the same experiments (Meijer et al. (1997) Plant J 11: 263-276; Meijer et al. (2000) supra).
Several class I HDZip proteins have been shown to be involved in light response and in abscisic acid (ABA)/water deficit related response (Hjellström et al. (2003) Plant Cell Environ 26: 1127-1136). Transgenic Arabidopsis overexpressing class I HDZip ATHB1, -3, -13, -20, and -23 suggest that these genes are involved in the regulation of cotyledon and leaf development (Aoyama et al. (1995) Plant Cell 7: 1773-1785; Hanson (2000) In Comprehensive summaries of Uppsala Dissertations from the Faculty of Science and Technology, Uppsala). The ATHB3, -13, -20, and -23 genes are similar and form a distinct subclass within the class I HDZip. Since these genes cause similar alterations in cotyledon shape when expressed constitutively, they are referred to as the pointed cotyledon (POC) HDZip genes. Hanson concludes that class I HDZip proteins that are closely related phylogenetically are also functionally related, in most cases.
Surprisingly, it has now been found that modulating expression in a plant a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof gives plants having increased yield under reduced nutrient availability, relative to corresponding wild type plants.
According to one embodiment of the present invention, there is provided a method for increasing yield in plants grown under reduced nutrient availability, relative to corresponding wild type plants, comprising modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof.
The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. A “control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Advantageously, performance of the methods according to the present invention results in plants having increased yield when grown under reduced nutrient availability, relative to corresponding wild type plants.
The term “increased yield” as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of a plant, aboveground (harvestable) parts, or increased root biomass, increased root volume, increased root number, increased root diameter or increased root length (of thick or thin roots), or increased biomass of any other harvestable part; (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of flowers (florets) per panicle, which is expressed as a ratio of number of filled seeds over number of primary panicles; (iv) increased seed fill rate; (v) number of (filled) seeds; (vi) increased seed size, which may also influence the composition of seeds; (vii) increased seed volume, which may also influence the composition of seeds (including oil, protein and carbohydrate total content and composition); (viii) increased (individual or average) seed area; (ix) increased (individual or average) seed length; (x) increased (individual or average) seed width; (xi) increased (individual or average) seed perimeter; (xii) increased harvest index (HI), which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; and (xiii) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight. An increased TKW may result from an increase in embryo size and/or endosperm size.
Preferably, the increased yield is selected from one or more of the following: increased total number of seeds, increased number of filled seeds, increased total seed yield, increased number of flowers per panicle, increased seed fill rate, increased HI, increased TKW, increased root length or increased root diameter, each relative to corresponding wild type plants.
Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under reduced nitrogen availability, relative to corresponding wild type plants, which method comprises modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof.
Taking corn as an example, an increased yield may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, among others. Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight, among others. An increased yield may also result in modified architecture, or may occur as a result of modified architecture.
Since the transgenic plants according to the present invention have increased yield under reduced nitrogen availability, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants or control plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including roots or seeds), or may be throughout substantially the whole plant. A plant having an increased growth rate may even exhibit early flowering. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants grown under reduced nitrogen availability, relative to corresponding wild type plants, which method comprises modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof.
Increased yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to corresponding wild type plants grown under comparable conditions. Plant with optimal growth conditions (grown under non-stress conditions) typically yield in increasing order of preference at least 90%, 87%, 85%, 83%, 80%, 77% or 75% of the average production of such a plant in a given environment. Average production of such plant may be calculated on harvest and/or season and/or location basis. Persons skilled in the art are aware of average yield productions of a crop. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses (as used herein) are the everyday abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures, excess or reduced availability of nutrients (macroelements and/or microelements). The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically stresses caused by pathogens, such as bacteria, viruses, fungi, nematodes, and insects. The term “non-stress” conditions as used herein are those environmental conditions that allow optimal growth of plants.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild abiotic stress conditions having increased yield relative to corresponding wild type plants. Therefore, according to the present invention, there is provided a method for increasing yield of plants grown under non-stress conditions or under mild abiotic stress conditions, relative to corresponding wild type, which method comprises modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. Preferably the mild abiotic stress conditions are reduced availability of nutrients.
Performance of the methods according to the present invention results in plants having increased tolerance to abiotic stress. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturation of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest.
Diverse environmental stresses activate similar pathways, as exemplified in the present invention for plants grown in drought stress and salt stress conditions. These examples should be seen as a screen to indicate the involvement of class I HDZip hox5 polypeptides or homologues thereof in increasing tolerance to abiotic stresses in general. A particularly high degree of “cross talk” is reported between drought stress and high-salinity stress (Rabbani et al. (2003) Plant Physiol 133: 1755-1767). Therefore, it would be apparent that a class I HDZip hox5 polypeptide or a homologue thereof would, along with its usefulness in increasing drought-tolerance and salt-tolerance in plants, also find use in protecting the plant against various other abiotic stresses.
The term “abiotic stress” as defined herein is taken to mean any one or more of: water stress (due to drought or excess water), anaerobic stress, salt stress, temperature stress (due to hot, cold or freezing temperatures), chemical toxicity stress and oxidative stress. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress. The term salt stress is not restricted to stress resulting from excess common salt (NaCl), but may be from one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
Another example of abiotic environmental stress is the reduced availability of one or more nutrients that need to be assimilated by the plants for growth and development. Because of the strong influence of nutrition utilisation efficiency on plant yield and product quality, a huge amount of fertilizer is poured onto fields to optimize plant growth and quality. Productivity of plants ordinarily is limited by three primary nutrients, phosphorous, potassium and nitrogen, which is usually the rate-limiting element in plant growth of these three. Therefore the major nutritional element required for plant growth is nitrogen (N). It is a constituent of numerous important compounds found in living cells, including amino acids, proteins (enzymes), nucleic acids, and chlorophyll. 1.5% to 2% of plant dry matter is nitrogen and approximately 16% of total plant protein. Thus, nitrogen availability is a major limiting factor for crop plant growth and production (Frink et al. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well a major impact on protein accumulation and amino acid composition. Therefore, of great interest are crop plants with increased yield when grown under nutrient limiting conditions, preferably nitrogen-limiting conditions.
Performance of the methods of the invention gives plants having increased yield when grown under abiotic stress conditions, relative to corresponding wild type. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under abiotic stress conditions, relative to corresponding wild type plants, which method comprises modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from one or more of the following: water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress. Alternatively or additionally, the abiotic stress is reduced nutrient availability. Preferably, the abiotic stress is reduced nitrogen availability.
Increased tolerance to abiotic stress is manifested by plants with increased yield, relative to corresponding wild type plants. In particular, such increased yield may include one or more of the following: increased total number of seeds, increased number of filled seeds, increased total seed yield, increased number of flowers per panicle, increased seed fill rate, increased HI, increased TKW, increased root length or increased root diameter, each relative to corresponding wild type plants. Preferably the increased tolerance to abiotic stress is increased tolerance to reduced nutrient availability, more preferably increased tolerance to reduced nitrogen availability.
Advantageously, performance of the methods of the invention gives plants having an increased greenness index under reduced nutrient availability relative to corresponding wild type plants. The greenness index is calculated from the digital images of plants. For each pixel belonging to the plant object on the image, the ratio of the green value versus the red value (in the RGB model for encoding color) is calculated. The greenness index as defined herein is expressed as the percentage of pixels for which the green-to-red ratio exceeds a given threshold. An increased greenness index may indicate reduced or delayed senescence which in turn allows prolongation of the photosynthetic activity of a plant, which in turn leads to various beneficial effects well known in the art.
Performance of the methods of the invention gives plants having an increased greenness index under reduced nutrient availability, relative to corresponding wild type plants. Therefore, according to the present invention, there is provided a method for increasing greenness index in plants grown under reduced nutrient availability relative to corresponding wild type plants, which method comprises modulating expression in a plant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. Preferably, the reduced nutrient availability conditions are reduced nitrogen availability conditions.
Rabbani et al. (2003, Plant Physiol 133: 1755-1767) report that similar molecular mechanisms of stress tolerance and responses exist between dicots and monocots. The methods of the invention are therefore advantageously applicable to any plant.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid sequence of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid sequence of interest. Therefore, the term “plant” as used herein encompasses a plant, plant part (including seeds), or plant cell obtainable by the methods of the invention, wherein each of the aforementioned comprises a recombinant nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant is a monocotyledonous plant, such as sugar cane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, triticale, millet, rye, sorghum or oats.
The term “class I HDZip hox5 polypeptide or homologue thereof” as defined herein refers to a polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads.
Additionally, the class I HDZip hox5 polypeptide or a homologue thereof may comprise any one or both of the following: (a) a Trp tail; and (b) the RPFF amino acid motif, where R is Arg, P Pro and F Phe. The motif of (b) precedes the acidic box, when examining the protein from N-terminal to C-terminal.
An example of a class I HDZip hox5 polypeptide as defined hereinabove comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads; and additionally comprising: (a) a Trp tail; and (b) the RPFF amino acid motif, where R is Arg, P Pro and F Phe, is represented as in SEQ ID NO: 2. Further such examples are given in Table A of Example 1 herein.
A class I HDZip hox5 polypeptide or homologue thereof is encoded by a class I HDZip hox5 gene/nucleic acid sequence. Therefore the term “class I HDZip hox5 gene/nucleic acid sequence” as defined herein is any gene/nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof as defined hereinabove.
Class I HDZip hox5 polypeptides or homologues thereof may readily be identified using routine techniques well known in the art, such as by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologues of class I HDZip hox5 polypeptides comprising a class I homeodomain and a leucine zipper with more than 5 heptads may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art (see Example 2 and FIG. 2 herein).
The various structural domains in a class I HDZip hox5 polypeptide, such as the homeodomain and the leucine zipper, may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucl Acids Res 30, 242-244; hosted by EMBL at Heidleberg), InterPro (Mulder et al., (2003) Nucl Acids Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucl Acids Res 30(1):276-280 (2002)). Leucine zipper prediction and heptad identification may be done using specialised software such as 2ZIP, which combines a standard coiled coil prediction algorithm with an approximate search for the characteristic leucine repeat (Bornberg-Bauer et al. (1998) Computational Approaches to Identify Leucine Zippers, Nucl Acids Res, 26(11): 2740-2746). Results of domain identification in class I HDZip hox5 polypeptide sequences are presented in Example 4 of this application.
Furthermore, the presence of an acidic box may also readily be identified. Primary amino acid composition (in %) to determine if a polypeptide domain is rich in specific amino acids may be calculated using software programs from the ExPASy server, in particular the ProtParam tool (Gasteiger E et al. (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784-3788). The composition of the protein of interest may then be compared to the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank. Within this databank, the average Asp (D) and Glu (E) content are of 5.3% and of 6.6% respectively, the combined average being of 11.9%. As an example, the acidic box of SEQ ID NO: 2 comprises 9.1% of D and 54.5% of E, the combined average being of 63.6% (see Example 4 herein). As defined herein, an acidic rich box has a combined Asp (D) and Glu (E) content (in % terms) above that found in the average amino acid composition (in % terms) of the proteins in the Swiss-Prot Protein Sequence database. An acidic box may be part of a transcription activation domain. Eukaryotic transcription activation domains have been classified according to their amino acid content, and major categories include acidic, glutamine-rich and proline-rich activation domains (Rutherford et al. (2005) Plant J. 43(5): 769-88, and references therein).
A selected number of polypeptides amongst the class I HDZip hox5 polypeptides or homologues thereof further comprise the RPFF amino acid motif, where R is Arg, P Pro and F Phe, This motif precedes the acidic box, when examining the protein from N-terminal to C-terminal (see FIG. 2). The presence of the RPFF may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called “expect” value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified.
A selected number of polypeptides amongst the class I HDZip hox5 polypeptides or homologues thereof may further comprise a Trp tail. A Trp tail as defined herein is the last 10 amino acids of the C-terminal of the polypeptide comprising at least one Trp residue (see FIG. 2).
Examples of class I HDZip hox5 polypeptides or homologues thereof (encoded by polynucleotide sequence accession number in parenthesis) are given in Table A of the Examples.
It is to be understood that sequences falling under the definition of “class I HDZip hox5 polypeptide or homologue thereof” are not to be limited to the sequences given in Table A, but that any polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads, may be suitable for use in performance of the methods of the invention.
Class I HDZip hox5 polypeptides or homologues thereof have DNA binding activity, preferably to 5 bp half-sites that overlap at a central position, CAA(A/T)ATTG, as detected in yeast one-hybrid assays (Meijer et al. (2000) Mol Gen Genet 263:12-21). In transient assays on rice cell suspensions, co-bombardement of a class I HDZip hox5 polypeptide with the GUS reporter gene resulted in an increase number of stained spots, which were also more intense in color (Meijer et al, supra). This assay is useful to demonstrate the activator function of class I HDZip hox5 polypeptides or homologues.
Examples of class I HDZip hox5 nucleic acid sequences include but are not limited to those listed in Table A of the Examples. Class I HDZip hox5 genes/nucleic acid sequences and variants thereof may be suitable in practising the methods of the invention. Variants of class I HDZip hox5 genes/nucleic acid sequences include portions of a class I HDZip hox5 gene/nucleic acid sequence and/or nucleic acid sequences capable of hybridising with a class I HDZip hox5 gene/nucleic acid sequence.
The term portion as defined herein refers to a piece of DNA encoding a polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads. A portion may be prepared, for example, by making one or more deletions to a class I HDZip hox5 nucleic acid sequence. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the class I HDZip hox5 portion. Preferably, the portion is a portion of a nucleic acid sequence as represented by any one of the nucleic acid sequences listed in Table A of Example 1 herein. Most preferably the portion is a portion of a nucleic acid sequence as represented by SEQ ID NO: 1.
Another variant of a class I HDZip hox5 gene/nucleic acid sequence is a nucleic acid sequence capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a class I HDZip hox5 gene/nucleic acid sequence as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads. Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid sequence as represented by any one of the nucleic acid sequences listed in Table A of Example 1 herein, or to a portion of any of the aforementioned sequences as defined hereinabove. Most preferably the hybridising sequence is one that is capable of hybridising to a nucleic acid sequence as represented by SEQ ID NO: 1.
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acid molecules are in solution. The hybridisation process can also occur with one of the complementary nucleic acid molecules immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acid molecules immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid sequence arrays or microarrays or as nucleic acid sequence chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acid molecules. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition.
“Stringent hybridisation conditions” and “stringent hybridisation wash conditions” in the context of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and are different under different environmental parameters. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either maintain or change the stringency conditions.
The Tm, is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M. Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45° C., though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1° C. per % base mismatch. The Tm, may be calculated using the following equations, depending on the types of hybrids:    1. DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):            Tm=81.5° C.+16.6xlog[Na+]a+0.41x %[G/Cb]−500x[Lc]−1−0.61x % formamide            2. DNA-RNA or RNA-RNA hybrids:            Tm=79.8+18.5 (log10[Na+]a)+0.58 (% G/Cb)+11.8 (% G/Cb)2−820/Lc             3. oligo-DNA or oligo-RNAd hybrids:            For <20 nucleotides: Tm=2 (/n)        For 20-35 nucleotides: Tm=22+1.46 (/n)            a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.    b only accurate for % GC in the 30% to 75% range.    c L=length of duplex in base pairs.    d Oligo, oligonucleotide; /n, effective length of primer=2×(no. of G/C)+(no. of A/T).Note: for each 1% formamide, the Tm, is reduced by about 0.6 to 0.7° C., while the presence of 6 M urea reduces the Tm, by about 30° C.
Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid sequence hybridisation assays or gene amplification detection procedures are as set forth above. Conditions of greater or less stringency may also be selected. Generally, low stringency conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with RNase. Examples of hybridisation and wash conditions are listed in Table 1 below.
TABLE 1Examples of hybridisation and wash conditionsWashStringencyPolynucleotideHybridHybridisationTemperatureConditionHybrid±Length (bp)‡Temperature and Buffer†and Buffer†ADNA:DNA> or65° C. 1 × SSC; or 42° C.,65° C.; 0.3 × SSCequal to 501 × SSC and 50% formamideBDNA:DNA<50Tb*; 1 × SSCTb*; 1 × SSCCDNA:RNA> or67° C. 1 × SSC; or 45° C.,67° C.; 0.3 × SSCequal to 501 × SSC and 50% formamideDDNA:RNA<50Td*; 1 × SSCTd*; 1 × SSCERNA:RNA> or70° C. 1 × SSC; or 50° C.,70° C.; 0.3 × SSCequal to 501 × SSC and 50% formamideFRNA:RNA<50Tf*; 1 × SSCTf*; 1 × SSCGDNA:DNA> or65° C. 4 × SSC; or 45° C.,65° C.; 1 × SSCequal to 504 × SSC and 50% formamideHDNA:DNA<50Th*; 4 × SSCTh*; 4 × SSCIDNA:RNA> or67° C. 4 × SSC; or 45° C.,67° C.; 1 × SSCequal to 504 × SSC and 50% formamideJDNA:RNA<50Tj*; 4 × SSCTj*; 4 × SSCKRNA:RNA> or70° C. 4 × SSC; or 40° C.,67° C.; 1 × SSCequal to 506 × SSC and 50% formamideLRNA:RNA<50Tl*; 2 × SSCTl*; 2 × SSCMDNA:DNA> or50° C. 4 × SSC; or 40° C.,50° C.; 2 × SSCequal to 506 × SSC and 50% formamideNDNA:DNA<50Tn*; 6 × SSCTn*; 6 × SSCODNA:RNA> or55° C. 4 × SSC; or 42° C.,55° C.; 2 × SSCequal to 506 × SSC and 50% formamidePDNA:RNA<50Tp*; 6 × SSCTp*; 6 × SSCQRNA:RNA> or60° C. 4 × SSC; or 45° C.,60° C.; 2 × SSCequal to 506 × SSC and 50% formamideRRNA:RNA<50Tr*; 4 × SSCTr*; 4 × SSC‡The “hybrid length” is the anticipated length for the hybridising nucleic acid sequence. When nucleic acid sequences of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.†SSPE (1 × SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH7.4) may be substituted for SSC (1 × SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridisation and wash buffers; washes are performed for 15 minutes after hybridisation is complete. The hybridisations and washes may additionally include 5 × Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.*Tb-Tr: The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids; the Tm is determined according to the above-mentioned equations.±The present invention also encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified nucleic acid.
For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
The class I HDZip hox5 nucleic acid sequence may be derived from any natural or artificial source. The gene/nucleic acid sequence may be isolated from a microbial source, such as yeast or fungi, or from a plant, algae or animal (including human) source. This nucleic acid sequence may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid sequence is of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. Preferably, the nucleic acid sequence may be isolated from a monocotyledonous species, further preferably from the family Poaceae, more preferably from Oryza genus, most preferably from Oryza sativa. More preferably, the class I HDZip hox5 nucleic acid sequence isolated from Oryza sativa is represented by SEQ ID NO: 1 and the class I HDZip hox5 polypeptide sequence is as represented by SEQ ID NO: 2.
The expression of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof may be modulated by introducing a genetic modification, by any one (or more) of the following methods: T-DNA activation, TILLING, site-directed mutagenesis, directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. Following introduction of the genetic modification, there follows a step of selecting for modulated expression of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof, which modulation in expression gives plants having increased yield under reduced nutrient availability, relative to corresponding wild type plants.
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353) involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or down stream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to overexpression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to overexpression of genes close to the introduced promoter. The promoter to be introduced may be any promoter capable of directing expression of a gene in the desired organism, in this case a plant. For example, constitutive, tissue-preferred, cell type-preferred and inducible promoters are all suitable for use in T-DNA activation.
A genetic modification may also be introduced in the locus of a class I HDZip hox5 gene using the technique of TILLING (Targeted Induced Local Lesions In Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a class I HDZip hox5 nucleic acid sequence capable of exhibiting class I HDZip hox5 activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher class I HDZip hox5 activity than that exhibited by the gene in its natural form. TILLNG combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei GP and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).
Homologous recombination allows introduction in a genome of a selected nucleic acid sequence at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offring a et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8). The nucleic acid sequence to be targeted (which may be a class I HDZip hox5 nucleic acid sequence or variant thereof as hereinbefore defined) need not be targeted to the locus of a class I HDZip hox5 gene, but may be introduced in, for example, regions of high expression. The nucleic acid sequence to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
Site-directed mutagenesis may be used to generate variants of class I HDZip hox5 nucleic acid sequences. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (Current Protocols in Molecular Biology, Wiley Eds).
Directed evolution may also be used to generate variants of class I HDZip hox5 nucleic acid sequences. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of class I HDZip hox5 nucleic acid sequences or portions thereof encoding class I HDZip hox5 polypeptides or homologues or portions thereof having an modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
T-DNA activation, TILLING, homologous recombination, site-directed mutagenesis, and directed evolution are methods to introduce a genetic modification to modulate expression of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. Therefore, according to the present invention, there is provided a method for modulating expression of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof, comprising introducing a genetic modification by one or more of: T-DNA activation, TILLING, homologous recombination, site-directed mutagenesis, and directed evolution.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a class I HDZip hox5 gene) is to introduce and express in a plant a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof. A class I HDZip hox5 polypeptide or a homologue thereof is defined as polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads. The nucleic acid sequence to be introduced into a plant may be a full-length nucleic acid sequence or may be a portion or a hybridising sequence as hereinbefore defined.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheetstructures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 2 below). The homologues useful in the methods according to the invention are preferably class I HDZip hox5 polypeptides as defined herein above.
Also encompassed by the term “homologues” are two special forms of homology, which include orthologous sequences and paralogous sequences, which encompass evolutionary concepts used to describe ancestral relationships of genes. The term “paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes. The term “orthologous” relates to homologous genes in different organisms due to speciation.
Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting the sequence in question (for example, SEQ ID NO: 1 or SEQ ID NO: 2) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX may be used when starting from nucleotide sequence, or BLASTP or TBLASTN when starting from the polypeptide, with standard default values. The BLAST results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then BLASTed back (second BLAST) against the sequences of the organism from which the sequence in question is derived. The results of the first and second BLASTs are then compared. When the results of the second BLAST give as hits with the highest similarity a class I HDZip hox5 nucleic acid sequence or class I HDZip hox5 polypeptide, then a paralogue has been found, if it originates from the same organism as for the sequence used in the first BLAST. In case it originates from an organism other than that of the sequence used in the first BLAST, then an orthologue has been found. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize the clustering. Preferably, such class I HDZip hox5 polypeptides have in increasing order of preference at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity or similarity (functional identity) to an unmodified class I HDZip hox5 polypeptide (preferably SEQ ID NO: 2; see Example 3 herein). Percentage identity between class I HDZip hox5 homologues outside of the homeodomain and the leucine zipper is reputedly low (see Example 3 herein). Examples of orthologs and paralogs of a class I HDZip hox5 polypeptide as represented by SEQ ID NO: 2 may be found in Table of Example 1 herein.
A homologue may be in the form of a “substitutional variant” of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions. Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions.
TABLE 2Examples of conserved amino acid substitutionsConservativeResidueSubstitutionsAlaSerArgLysAsnGln; HisAspGluGlnAsnCysSerGluAspGlyProHisAsn; GlnIleLeu, ValLeuIle; ValLysArg; GlnMetLeu; IlePheMet; Leu; TyrSerThr; GlyThrSer; ValTrpTyrTyrTrp; PheValIle; Leu
A homologue may also be in the form of an “insertional variant” of a protein, i.e. where one or more amino acid residues are introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
Homologues in the form of “deletion variants” of a protein are characterised by the removal of one or more amino acids from a protein.
Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The class I HDZip hox5 polypeptide or homologue thereof may be a derivative. “Derivatives” include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
The class I HDZip hox5 polypeptide or homologue thereof may be encoded by an alternative splice variant of a class I HDZip hox5 gene/nucleic acid sequence. The term “alternative splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art. Preferred splice variants are splice variants of a nucleic acid sequence encoding a polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads. Additionally, a class I HDZip hox5 polypeptide or a homologue thereof may comprise one or both of the following: (a) a Trp tail; and (b) the RPFF amino acid motif, where R is Arg, P Pro and F Phe. The motif of (b) precedes the acidic box, when examining the protein from N-terminal to C-terminal. Further preferred are splice variants of nucleic acid sequences as listed in Table A of Example 1 herein. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 1.
The homologue may also be encoded by an allelic variant of a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or a homologue thereof, preferably an allelic variant of a nucleic acid sequence encoding a polypeptide comprising from N-terminal to C-terminal: (i) an acidic box; and (ii) a class I homeodomain; and (iii) a leucine zipper with more than 5 heptads. Additionally, a class I HDZip hox5 polypeptide or a homologue thereof may comprise one or both of the following: (a) a Trp tail; and (b) the RPFF amino acid motif, where R is Arg, P Pro and F Phe. The motif of (b) precedes the acidic box, when examining the protein from N-terminal to C-terminal. Further preferred are allelic variants of nucleic acid sequences listed in Table A of Example 1 herein. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
According to a preferred aspect of the present invention, modulated expression of the class I HDZip hox5 nucleic acid sequence is envisaged. Methods for modulating expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acid sequences which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a class I HDZip hox5 nucleic acid sequence. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. Methods for reducing the expression of genes or gene products are well documented in the art.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:                (i) A class I HDZip hox5 nucleic acid sequence, as defined hereinabove;        (ii) One or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally        (iii) A transcription termination sequence.        
Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid sequence encoding a class I HDZip hox5 polypeptide or homologue thereof). The sequence of interest is operably linked to one or more control sequences (at least to a promoter). The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid sequence molecule in a cell, tissue or organ. The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. An example of an inducible promoter being a stress-inducible promoter, i.e. a promoter activated when a plant is exposed to various stress conditions. Additionally or alternatively, the promoter may be a tissue-preferred promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc. Promoters able to initiate transcription in certain tissues only are referred to herein as “tissue-specific”.
In one embodiment, a class I HDZip hox5 nucleic acid sequence is operably linked to a constitutive promoter. A constitutive promoter is transcriptionally active during most but not necessarily all phases of its growth and development and is substantially ubiquitously expressed. The constitutive promoter is preferably a GOS2 promoter, more preferably the constitutive promoter is a rice GOS2 promoter, further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 33 or SEQ ID NO: 52, most preferably the constitutive promoter is as represented by SEQ ID NO: 33 or SEQ ID NO: 52. It should be clear that the applicability of the present invention is not restricted to the class I HDZip hox5 nucleic acid sequence represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a class I HDZip hox5 nucleic acid sequence when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used perform the methods of the invention are shown in Table 3 below.
TABLE 3Examples of constitutive promotersGeneExpressionSourcePatternReferenceActinConstitutiveMcElroy et al., Plant Cell, 2: 163-171, 1990CAMV 35SConstitutiveOdell et al., Nature, 313: 810-812, 1985CaMV 19SConstitutiveNilsson et al., Physiol. Plant. 100: 456-462,1997GOS2Constitutivede Pater et al., Plant J Nov; 2(6): 837-44,1992UbiquitinConstitutiveChristensen et al., Plant Mol. Biol. 18:675-689, 1992RiceConstitutiveBuchholz et al., Plant Mol Biol. 25(5):cyclophilin837-43, 1994Maize H3ConstitutiveLepetit et al., Mol. Gen. Genet. 231:histone276-285, 1992Actin 2ConstitutiveAn et al., Plant J. 10(1); 107-121, 1996HMGBConstitutiveWO 2004/070039
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
The genetic construct may optionally comprise a selectable marker gene. As used herein, the term “selectable marker gene” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid sequence construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source). Expression of visual marker genes results in the formation of colour (for example β-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
In a preferred embodiment, there is provided a gene construct comprising:                (i) A class I HDZip hox5 nucleic acid sequence, as defined hereinabove;        (ii) A constitutive promoter capable of driving expression of the nucleic acid sequence of (i); and optionally        (iii) A transcription termination sequence.        
The constitutive promoter is preferably a GOS2 promoter, more preferably the constitutive promoter is the rice GOS2 promoter, further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 33 or to SEQ ID NO: 52, most preferably the constitutive promoter is as represented by SEQ ID NO: 33 or to SEQ ID NO: 52. The invention further provides use of a construct as defined hereinabove in the methods of the invention.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts or plant cells thereof obtainable by the method according to the present invention, which plants or parts or cells thereof comprise a transgene class I HDZip hox5 nucleic acid sequence.
The invention also provides a method for the production of transgenic plants having increased yield under reduced variant nutrient availability, relative to corresponding wild type plants, comprising introduction and expression in a plant of a nucleic acid sequence encoding class I HDZip hox5 polypeptide or a homologue thereof.
More specifically, the present invention provides a method for the production of transgenic plants having increased yield under reduced nutrient availability, relative to corresponding wild type plants, which method comprises:                (i) introducing and expressing in a plant, plant part or plant cell a class I HDZip hox5 nucleic acid sequence; and        (ii) cultivating the plant cell under conditions promoting plant growth and development.        
The nucleic acid sequence may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid sequence is preferably introduced into a plant by transformation.
The term “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al. (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al. (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al. (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing a class I HDZip hox5 gene/nucleic acid sequence are preferably produced via Agrobacterium-mediated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also includes host cells containing an isolated class I HDZip hox5 nucleic acid sequence. Preferred host cells according to the invention are plant cells.
The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of class I HDZip hox5 nucleic acid and use of class I HDZip hox5 polypeptides or homologues thereof. Such uses relate to increasing yield in plants grown under reduced nutrient availability, relative to wild type plants, as defined hereinabove in the methods of the invention. Preferably, the increased yield is one or more of: increased total seed yield per plant, increased number of filled seeds, increased seed fill rate, increased number of flowers per panicle, or increased harvest index.
Class I HDZip hox5 nucleic acid sequences or variants thereof, or class I HDZip hox5 polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a class I HDZip hox5 gene or variant thereof. The class I HDZip hox5 genes/nucleic acid sequences or variants thereof, or class I HDZip hox5 polypeptides or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield when grown under reduced nutrient availability, as defined hereinabove in the methods of the invention. The class I HDZip hox5 gene may, for example, be a nucleic acid sequence as listed in Table A of Example 1 herein.
Allelic variants of a class I HDZip hox5 gene/nucleic acid sequence may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give plants with increased yield under reduced nutrient availability, relative to corresponding wild type plants. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of any one of the nucleic acid sequences listed in Table A of Example 1 herein. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
A class I HDZip hox5 nucleic acid sequence or variant thereof may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of class I HDZip hox5 nucleic acid sequences or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The class I HDZip hox5 nucleic acid sequences or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the class I HDZip hox5 nucleic acid sequences or variants thereof. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acid sequences may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the class I HDZip hox5 nucleic acid sequence or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid sequence probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid sequence probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid sequence amplification-based methods for genetic and physical mapping may be carried out using the nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic acid sequence Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic acid sequence Res. 17:6795-6807). For these methods, the sequence of a nucleic acid sequence is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
The methods according to the present invention result in plants having increased yield under reduced nutrient availability, as described hereinbefore. This increased yield may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.